Thiarubrine antifungal agents

ABSTRACT

This invention pertains to a novel group of Thiarubrine substances and closely related derivatives, useful as antifungal and antibiotic agents.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No. 07/447,893, filed Dec. 7, 1989, which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention pertains to a novel group of Thiarubrine substances and closely related derivatives that are useful as antifungal and antibiotic agents. The invention further pertains to a novel method for isolating the Thiarubrine substances.

BACKGROUND OF THE INVENTION

A publication entitled "Two dithiacyclohexadiene Polyacetylenes from Chaenactis douglasii and Eriophvllum lanatum", Norton, R.A.; Finlayson, A.J.; Towers, G.H.N.; Phytochemistry, 24(2), 356-7 (1985) discloses two dithiapolyacetylenes isolated from the roots of C. douglasii and from the plant roots as well as root cultures of E. lanatum. The two dithiapolyacetylenes have the respective chemical names 3 - (1- -propynyl) 6 (5-hexen-3-yn-1-ynyl)1,2-dithiacyclohexa-3,5- diene and 3 (pent-3-yn-1-ynyl) 6 (3-buten-1-ynyl)-1,2- dithiacyclohexa-3,5-diene. The trivial names Thiarubrine A and Thiarubrine B, respectively, have been given to these compounds.

Another publication entitled "Production of Antibiotic Thiarubrines by a Crown Gall Tumor Line of Chaenactis douglasii", Cosio, E.G.; Norton, R.A.; Towers, E.; Finlayson, A.J.; Rodriguez, E.; Towers, G.H.N.; J. Plant Physiol., 124 (1-2), 155-64 (1986) discloses dithiacyclohexadiene polyacetylenes which display considerable antimicrobial activity. A culture that accumulates these compounds was obtained by selection of red-colored areas from crown gall tumor cultures of Chaenactis douglasii. The tumors were induced by Agrobacterium tumefaciens strain A277. The main acetylenic products found were two Thiarubrines, A and B, and their corresponding thiophenes. Average yields of thiarubrines and thiophenes were 2.5 and 0.24 mg/.g dry weight, respectively, which are similar to the values seen in intact plants. The products accumulate in intercellular spaces arranged around a core of vascular tissue, forming red nodules of varying size. The structures crudely resemble the tissue organization of the roots of this plant, the only organ where these compounds accumulate. Formation of rootlets, however, did not take place at any stage during the growth of the tumors. Apparently, accumulation of these polyacetylenes by the tumor line is not a direct result of cellular transformation but a secondary effect of the existing degree of tissue differentiation.

The publication "Thiarubrine Accumulation in Hairy Root Cultures of Chaenactis douglasii", Constabel, C.P.; Towers, G.H.N.; J. Plant Physiol., 133(1), 67-72 (1988) discloses hairy root cultures of C. douglasii established using Agrobacterium rhizogenes strain TR7. One culture line accumulated twice the levels of the antifungal polyines, Thiarubrines A and B, compared to non-transformed control root cultures, while maintaining rapid growth. The combination of fast growth and high thiarubrine accumulation could not be duplicated in controls by adding exogenous NAA to the culture medium. Hairy root cultures also produced less Thiarubrine B relative to Thiarubrine A compared to controls.

A publication entitled "Antiviral Properties of Thiarubrine A, a Naturally Occurring Polyine", Hudson J.B.; Graham E.A.; Fong R; Finlayson A.J., Towers G.H.N.; Planta Med 0(1), 1986, 51-54 relates to the naturally occurring polyine, Thiarubrine A. It was evaluated for its antiviral properties in the presence and absence of long wave UV radiation (UV-A). Four viruses and a mammalian cell line were used as targets. The two mammalian viruses, murine cytomegalovirus and Sindbis virus, both of which possess membranes, were extremely sensitive to the compound, but only in the presence of UV-A radiation. The bacteriophage T4 was slightly affected in UV-A only, whereas the bacteriophage M13 was completely unaffected. Thus Thiarubrine A is photoactive against membrane containing viruses. In contrast mouse cells were moderately sensitive to the compound in the presence of UV-A, and somewhat less sensitive in the dark.

Thiarubrine A is also discussed in "Antibiotic Properties of Thiarubrine A a Naturally Occurring Dithiacyclohexadiene Polyine", Towers G.H.N.; Abramowski Z; Finlayson A.J.; Zucconi A; Planta Med 0 (3), 1985, 225-229. Thiarubrine A, a dithiacyclohexadiene polyine from the roots of Chaenactis douglassii, is disclosed as exhibiting strong antifungal activity towards Candida albicans and Aspergillus fumigatus at concentrations comparable to those of amphotericin B. It also exhibits cytotoxic activity towards Escherichia coli, Bacillus subtilis and Hycobacteria sp. and the nematode Coenorhabditis elogans. A thiophene derived from thiarubrine A was phototoxic, requiring UV-A light for biological activity. The effects of Thiarubrine A, thiophene A and alpha-terthienyl on the above organisms, as well as on CHO cells, were compared.

Polyacetylene groups and thiophenes are disclosed in "comparison of the antiviral effects of naturally occurring thiophenes and polyacetylenes", Hudson J. B.; Graham E.A.; Chan G; Finlayson A.J.; Towers G.H.N.; Planta Med 0 (6], 1986 (Recd. 1987), 453-457. Five naturally 35 occurring compounds, containing various thiophene and polyacetylene groups, were compared with respect to their phototoxic activities against two animal viruses, murine cytomegalovirus and Sindbis virus, both of which possess membranes. Alpha-Terthienyl was extremely toxic to both viruses, but only in the presence of long wave ultraviolet radiation. The order of potency was alpha-terthienyl thiarubrine-A > phenylheptatriyne > ACBP-thiophene > thiophene-A (hydrolysis product of thiarubrine A). The murine-CMV, which had been inactivated by any of these compounds, was still capable of penetrating cultured mouse cells efficiently and reaching the cell nucleus, the normal site of virus replication. The results are discussed in terms of possible mechanisms of action of phototoxic thiophenes and polyacetylenes.

Canadian Patent No. 1,169,767, issued Jun. 26, 1984, Towers et al., discloses cercaricidal compositions containing naturally occurring conjugated polyacetylenes and method for controlling cercariae using the same.

Canadian Patent No. 1,172,460, granted Aug. 14, 1984, Towers et al., discloses a method for controlling weeds using naturally occurring conjugated polyacetylenes.

Canadian Patent No. 1,173,743, granted Sep. 4, 1984, Towers et al., discloses a method for controlling pests using naturally occurring conjugated polyacetylenes.

SUMMARY OF THE INVENTION

The invention is directed to a novel substance having antifungal and anti-bacteria activity of the formula: ##STR1## wherein R₁ is CH₃ or CH₂ R₃ ;

R₂ is ##STR2## --CHR₃ --CH₂ R₃ ; --HCHa--CH₂ R₃ ; --CHR₃ --CH₂ Ha; CH═CH₂ ;

R₃ is H; OH; --CHO; COOH or COOR₄ wherein R₄ is derived from an alcohol;

Ha is F; Cl; Br; or I;

n is 1 or 2; and

m is 1 or 2, and pharmaceutically acceptable salts thereof, provided that when R₁ is CH₃, n is 1 and m is 2, then R₂ is not CH═CH₂ ; and provided that when R₁ is CH₃, n is 2 and m is 1, then R₂ is not CH═CH₂.

The invention is also directed to the bactericide or fungicide Thiarubrine D ##STR3## the fungicide Thiarubrine E

    (CH.sub.3 --C.tbd.C--R--(C.tbd.C).sub.2 --CHOH--CH.sub.2 OH);

the fungicide Thiarubrine F

    (CH.sub.3 --C.tbd.C--R--(C.tbd.C).sub.2 --CHCl--CH.sub.2 OH);

the fungicide Thiarubrine G

(CH₃ --C.tbd.C--R--(C.tbd.C)₂ --CHOH--CH₂ Cl);

and the fungicide Thiarubrine H

    (HOCH.sub.2 --C.tbd.C--R--(C.tbd.C).sub.2 --CH═CH.sub.2);

where R is ##STR4##

Bacteria may be treated with the Thiarubrine substance in association with light of a wavelength range about 320 to 400 nanometers, or specifically, in association with light of a wavelength about 350 nanometers.

The invention is also directed to an antifungal composition comprising a Thiarubrine substance of the formula: ##STR5## wherein R₁ is CH₃ or CH₂ R₃ ;

R₂ is ##STR6## --CHR₃ --CH₂ R₃ ; --HCHa--CH₂ R₃ ; --CHR₃ --CH₂ Ha; CH═CH₂ ;

R₃ is H; OH; --CHO; COOH or COOR₄ wherein R₄ is derived from an alcohol;

Ha is F; Cl; Br; or I;

n is 1 or 2; and

m is 1 or 2 and pharmaceutically acceptable derivatives thereof, provided that when R₁ is CH₃, n is 1 and m is 2, then R₂ is not CH═CH₂ ; and provided that when R₁ is CH₃, n is 2 and m is 1, then R₂ is not CH═CH₂ in association with a pharmaceutically acceptable carrier.

In the composition, Ha may be chlorine, R₃ may be OH, R₃ may be OH, n may be 1 and m may be 2. The carrier may be distilled water, saline or any other pharmaceutically acceptable carrier.

The composition can be used to control E. coli. Staphylococcus aureus, Streptococcus faecalis. Mycobacterium sp. or Enterobacter aerogenes in dark and more effectively in the presence of light having a wavelength of about 320 to 400 nanometers, or specifically, having a wavelength of about 350 nanometers. The composition can be used to control Candida albicans, Aspergillus fumigatus or Cryptococcus neoformans as well.

The foregoing Thiarubrine substance is present in the composition at a concentration of about 1 to 100 nanograms/ml of carrier, or specifically, at a concentration of about 10 nanograms/ml of carrier.

The invention is also directed to a method of controlling Candida albicans or Aspergillus which comprises exposing a host infected with the Candida albicans or Aspergillus to an antifungal effective amount of a compound of the formula: ##STR7## wherein R₁ is CH₃ or --CH₂₀ H; and R₂ is ##STR8## CHOH--CH₂ OH; --HCCl--CH₂ OH; CHOH--CH₂ Cl or CH═CH₂.

Candida albicans may be treated at a concentration of Thiarubrine to Candida albicans in the range of about 1.0 to about 100 nanograms/ml of distilled water.

The invention pertains to an antifungal composition comprising Thiarubrine D and a pharmaceutically acceptable carrier. The Thiarubrine D can be present in the composition at a concentration of 7 nanograms/ml of carrier. The carrier in the composition can be water or any other pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION Novel Thiarubines

The present invention is based on the discovery of a group of novel dithiacyclohexadiene compounds isolated from extracts of a common ragweed Ambrosia chamissonis Asteraceae (tribe Heliantheae) found in coastal areas ranging from southern California to British Columbia, Canada. The novel compounds, isolated from extracts of root tissues, have been found to possess remarkable antifungal and antibactericidal activity.

The compounds which are somewhat similar in structure to dithiacyclohexadienes, i.e., Thiarubrine A and B, previously isolated from members of the Asteraceae family including Chaenactis douglasii, A. chamissonis and other species have been generically designated by the coined name Thiarubrine.

The novel Thiarubrine substances, and their closely related homologues which have been discovered, can be represented by the following generic formula: ##STR9## wherein R₁ is CH₃ or CH₂ R₃ ;

R₂ is: ##STR10## --CHR₃ --CH₂ R₃ ; --HCHa--CH₂ R₃ ; --CHR₃ 1'CH₂ Ha; --CH═CH₂ ;

R₃ is H; OH; --CHO; COOH or COOR₄ wherein R₄ is derived from an alcohol;

Ha is F; Cl; Br; I;

n is 1 or 2; and

m is 1 or 2, provided that when R₁ is CH₃, n is 1 and m is 2, then R₂ is not CH═CH₂ ; and provided that when R₁ is CH₃, n is 2 and m is 1, then R₂ is not CH═CH₂.

A series of five novel substances have been chemically identified, assigned the generic trivial name Thiarubrine and have been given letter designations as follows:

1. Thiarubrine D: (CH₃ --C.tbd.C--R--(C.tbd.C)₂ ##STR11## ); 2. Thiarubrine E: (CH₃ --C.tbd.C--R--(C.tbd.C)₂ --CHOH--CH₂ OH);

3. Thiarubrine F: (CH₃ --C.tbd.C--R--(C.tbd.C)₂ --CHCl--CH₂ OH);

4. Thiarubrine G: (CH₃ --C.tbd.C--R--(C.tbd.C)₂ --CHOH--CH₂ Cl); and

5. Thiarubrine H: (HOCH₂ --C.tbd.C--R--(C.tbd.C)₂ --CH═CH₂);

wherein R is ##STR12##

Method for Preparing the Novel Thiarubrines

The therapeutically effective thiarubrine antifungal and antibacterial agents are prepared by a method comprising steps as follows:

(a) tissues of plant(s) of the Asteraceae family that contain thiarubrines, e.g., Ambrosia chamissonis (var.), including the whole plant, plant parts, or tissue cultures thereof, are rinsed with water and are either airdried, frozen, or stored in various organic solvents (i.e. hexane, methanol, etc.);

(b) the roots and or various plant parts (i.e. roots, leaves, stems, etc.) obtained as in (a) are either lyophilized directly or the root bark is peeled and/or lyophilized, or blended or used directly;

(c) the plant parts as in (a) or (b) are extracted by either percolation with various organic solvents (i.e. hexane, ethyl acetate, methanol, etc.), or by cold organic solvent extraction using various solvents (hexane, methanol, etc.) followed by filtration or centrifugation, or by supercritical fluid extraction (SFE) (i.e. carbon dioxide or ammonia as extractants);

(d) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, and subjected to gel filtration (e.g. on Sephadex) with water +water +miscible organic solvents as the mobile phase; or alternatively

(e) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, subjected to reversed-phase TLC, column or HPLC chromatography, using water, and/or water + water-miscible organic solvents, all with or without buffers, as the mobile phase; or alternatively,

(f) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, subjected to silica gel TLC, column or HPLC chromatography with various organic solvents (i.e. freon, low-boiling hydrocarbons, alone or in mixture with polar modifiers, e.g. diethyl ether, ethyl acetate, etc.) as the mobile phase; or alternatively

(g) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, subjected to SFE (supercritical fluid extraction) or SFC (supercritical fluid chromatography) with various gases alone or in mixture with polar modifiers (i.e. water, methanol, etc.); or alternatively

(h) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, subjected to counter-current partition chromatography using anhydrous biphasic systems (i.e. hexane/acetonitrile, etc); or alternatively

(i) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, and subjected to gas chromatography;

(j) the thiarubrine-containing fractions display a red or red/brown chromophoric band and are detected by uv or visible λ=200-550 nm) detectors;

(k) the thiarubrines are obtained by collecting, and concentrating the uv/vis active fractions as in step (d); or alternatively

(1) the thiarubrines are obtained by collecting, and concentrating the uv/vis active fractions as in step (e); or alternatively

(m) the thiarubrines are obtained by collecting, and concentrating the uv/vis active fractions as in step (f); or alternatively

(n) the thiarubrines are obtained by collecting, and concentrating the uv-vis active fractions as in step (g); or alternatively

(o) the thiarubrines are obtained by collecting, and concentrating the uv/vis active fractions as in step (h); or alternatively

(p) the thiarubrines are obtained by collecting, and concentrating the uv/vis active fractions as in step (i).

Applications of Thiarubrines

The novel Thiarubrine compounds of the invention display strong antifungal activity in the dark and strong antifungal and bactericidal activity in association with ultra-violet A light in the wave length range 320 to 400 nm, and notably 350 nm.

These substances are effective in killing strains of Candida albicans. Aspergillus fumigatus and Cryptococcus neoformans in the dark. They are effective in killing Escherichia coli, Staphylococcus aureus, Streptococcus faecalis, Mycobacterium sp, and Enterobacter aerogenes.

Thus, the Thiarubrine compounds are advantageously used for treatment of infections induced by the above mentioned fungal and bacterial agents. For example, the epoxide compound, designated Thiarubrine D, is a novel complex sulphur-containing substance. Thiarubrine D can be described by the specific formula: ##STR13##

Its very high antifungal activity in extremely low concentrations indicates a usefulness in vivo, i.e. for human patients.

The chemical, Thiarubrine D, is light sensitive and breaks down under light to the corresponding thiophene. It is therefore necessary to store the chemical in the dark and for effectiveness to conduct antifungal therapy in the absence of blue and ultraviolet light.

We have demonstrated in a series of tests which are tabulated below that Thiarubrines D, E, F, G and H will inhibit the growth of Candida albicans, at very low levels, i.e. in the nanogram/ml range.

The following Examples are presented to illustrate the present invention and in no way intended to be a limitation thereon.

EXAMPLES Preparation of Novel Thiarubrines

A. chamissonis were collected from Marin County, Calif. and from Tiaswwassen, British Columbia.

Freeze-dried roots (350-500 g) were extracted with cold MeOH, concentrated in vaccuo at 30° C. and redissolved in MeCN. The combined extracts were taken up in CHCl₃, dried and evaporated. The residue was chromatographed on a silica gel 60 column (Merck 70 -230 mesh), eluted with n-heptane-n-hexane (1:4) gradually changed to n-hexane-EtOAc (2:3) and finally EtOAc. Extracts and fractions for HPLC analysis were taken to dryness and resuspended in MeCN. Samples (20 and 100 μl) were injected on a Varian 5000 liquid chromatograph with a MCH-10 reverse phase column (4×300 mm) using MeCN-H₂ O (18:7) at a flow rate of 1.0 ml min ⁻¹. A much better resolution of Thiarubrines F and G and H was achieved with MeCN-H₂ O (2:3 and 1:1) and each compound isolated by extraction of the recovered HPLC fractions with CHCl₃. All procedures were performed in dim light to prevent photodegradation of the compounds.

MeCN

Thiarubrine D: UV λ_(max) nm: 484, 344, 272; MS m/z (rel. int.): 244 (M) +(100), 214, (75), 182 (21), 170 (98).

MeCN

Thiarubrine E: UV λ_(max) nm: 482, 342,326(sh): MS m/z (rel. int.): 262 (M) + (100), 231, (M-CH₂ OH) + (80), 199 (M-CH₂ OH-S) +(15).

MeCN

Thiarubrine F: UV-vis λ_(mes) nm: 484, 344, 230; MS m/z (rel. int.) 279.9783 (M) + (80), 249 [M-CH₂ OH]⁺ (30), 248 (M-S)⁺ (50), 217 [M-S-CH₂ OH]⁺, (48).

MeCN

Thiarubrine G: UV-vis-λ_(mes) nm: 484, 342, 228, MS m/z (rel. int.): 279.9780 (M)⁺, (100), 248 (M-S)⁺, (18) 244 (M-HCl)⁺ (50), 231, [M-CH₂ Cl]⁺ (98), 199 (M-CH₂ Cl-S)⁺ (30).

MeCN

Thiarubrine H: UV-vis λ_(mes) nm: 490, 354, 232, MS m/z (rel. int.): 244.0009 (M)⁺, (100), 227 (M-OH)⁺ (5), 212 (M-S)⁺ (65), 195 (M-S-OH)⁺ (6).

Biological Activity of Thiarubrines

The following experiments demonstrate the biological activity of the Thiarubrines of the present invention.

                  TABLE 1                                                          ______________________________________                                         Comparison of Concentrations of Thiarubrines and                               Fungizone Causing 50% Growth lnhibition (μg/ml)                             During 24 Hours Exposure                                                                Thiarubrines                                                                   A    D          E       Fungizone                                     ______________________________________                                         C. albicans                                                                               0.028   0.0006     0.0014                                                                              0.037                                       p815       0.035  0.12       0.024 >5.0                                        L1210      0.035   0.047     0.027 2.8                                         WEHl-3     0.027  0.05       0.04  1.9                                         3T3        0.45   0.48       0.12  >5.0                                        ______________________________________                                          P815 = Mouse mastocytoma                                                       L1210 = Mouse lymphocytic leukemia                                             WEHl3 = Mouse myelomonocyte                                                    3T3 = Mouse fibroblasts                                                  

                  TABLE 2                                                          ______________________________________                                         Toxicity Index of Thiarubrines and Fungizone                                   for Different Animal Cell Lines                                                            Thiarubrines                                                                   A    D        E     Fungizone                                      ______________________________________                                         LC.sub.50 P815                                                                               1      200      17  >135                                         LC.sub.50 C. albicans                                                          LC.sub.50 L1210                                                                              1       78      19    76                                         C. albicans                                                                    LC.sub.50 WEHI-3                                                                             1       83      28    51                                         LC.sub.50 C. albicans                                                          LC.sub.50 3T3 12     800      86  >135                                         LC.sub.50 C. albicans                                                          ______________________________________                                    

Table 2 shows how many times the concentrations needed to inhibit growth in 50% of animal cells are higher than doses required to inhibit growth in 50% of C. albicans.

                                      TABLE 3                                      __________________________________________________________________________     Comparison of Minimal Concentration Causing 100% Growth                        Inhibition (μg/ml) During 24 Hours Exposure                                          Thiarubrines                                                                               Thiophenes                                                         A   D   E   D  E   Fungizone                                                                            Gentamycin                                   __________________________________________________________________________     E. coli  0.625                                                                              0.078                                                                              0.019                                                                               5.0                                                                              >5.0      1.25                                         P. aeruginosa                                                                           2.5 0.625                                                                              0.625                                                                              >5.0                                                                              >5.0      2.5                                          C. albicans                                                                             0.16                                                                                0.0024                                                                            0.0048                                                                             >5.0                                                                              >5.0                                                                               0.16                                               P815 mastocytoma                                                                        0.625                                                                              1.25                                                                               0.3125                                                                             >5.0                                                                              >5.0                                                   __________________________________________________________________________      This table shows that minimal concentration causing 100% growth inhibitio      of animal tumourcells  P815 line is about 500 times greater than the           concentration needed for 100% growth inhibition of C. albicans in the cas      of Thiarubrine D, 65 times greater for Thiarubrine E, and about 4 times        for Thiarubrine A.                                                       

                                      TABLE 4                                      __________________________________________________________________________     Comparison of Minimal Concentration Causing 50% Growth                         Inhibition (μg/ml) During 24 Hours Exposure                                          Thiarubrines                                                                               Thiophenes                                                         A  D    E   D   E  Fungizone                                                                            Gentamycin                                   __________________________________________________________________________     E. coli  0.18                                                                              0.02 0.015                                                                              1.7 >5.0     0.37                                         P. aeruginosa                                                                           0.68                                                                              0.21 0.28                                                                               >5.0                                                                               >5.0     1.0                                          C. albicans                                                                             0.028                                                                              0.0006                                                                              0.0014                                                                            1.1  4.5                                                                              0.037                                              P815 mastocytoma                                                                        0.035                                                                             0.12 0.024                                                                              3.5  4.0                                                  __________________________________________________________________________      According to these data, concentration needed to inhibit growth of animal      cells  P815 in 50% is 200 times higher than for C. albicans in the case o      Thiarubrine D, 17 times higher for Thiarubrine E and the same for              Thiarubrine A.                                                                 Doses inhibiting growth in 50% were estimated by plotting concentration o      a logarithmic scale and percent of growth inhibition for 24 hours exposur      on a probability scale.                                                  

                  TABLE 5                                                          ______________________________________                                                                   Thiarubrine D                                                                  Minimal Inhibitory                                                    ATCC     Concentration                                        Organism         No.      (ng/ml)                                              ______________________________________                                          1. Staphylococcus aureus                                                                       25923       10,000                                             2. Streptococcus faecalis                                                                      29212       10,000                                             3. Escherichia coli                                                                            25992       1,000                                              4. Pseudomonas aeruginosa                                                                      27853       10,000                                             5. Staphylococcus aureus                                                                       29213       10,000                                             6. Enterobacter aerogenes                                                                      35029       10,000                                             7. Klebsiella pneumoniae                                                                       33495       10,000                                             8. Staphylococcus epidermis                                                                    29972       10,000                                             9. Cryptococcus neoformans                                                                     F548        64                                                10. Candida pseudotropicalis 10,000                                            11. Staphylococcus aureus    10,000                                               (metacylliu res.)                                                           12. Pseudomonas aeruginosa                                                                      141         >10,000                                           13. Pseudomonas aeruginosa                                                                      205         >10,000                                           14. Pseudomonas aeruginosa                                                                      3E5         10,000                                            15. Zanthomonas maltophilia                                                                     3E6         10,000                                            16. Staphylococcus aueras                                                                       B4287       10,000                                               (metacylliu res.)                                                           17. Acinetobacter                                                                               C42         1,000                                             18. Acinetobacter                                                                               6C10        1,000                                             19. Enterobacter cloacoe                                                                        1A5         1,000                                             20. Enterobacter cloacoe                                                                        9E5         1,000                                             21. Aspergillus  05          32                                                22. Candida      01          16                                                23. Candida      02          16                                                24. Candida      03          64                                                25. Candida      R4197       16                                                26. Enterobacter sp.                                                                            9E7         10,000                                            27. Serratia marscenes                                                                          3A3         10,000                                            28. Listeria monocytogenes                                                                      106         10,000                                            29. Listeria monocytogenes                                                                      301         10,000                                            30. Enterococcus faecalis                                                                       1A3         10,000                                            31. Klebsiella sp.           1,000                                             32. Candida      F617        8                                                 33. Enterococcus             10                                                   (gentamycin res.)                                                           34. Aspergillus  F604        64                                                35. Enterococcus faecalis                                                                       207         1,000                                             ______________________________________                                    

Results were obtained by agar dilution method. Mueller Hinton agar plates (pH - 7.4) with different doses of ThD were inoculated with 10⁴ cfu/spot and incubated 21 hours at 34°. MIC is the lowest concentration with no growth.

                  TABLE 6                                                          ______________________________________                                         Sensitivity of Aspergillus fumigatus                                           to Thiarubrines A. D. E.                                                                     Dose     Zone of Growth                                          Compound      (ng/disc)                                                                               Inhibition (mm)                                         ______________________________________                                         Thiarubrine A 10,000   27                                                                    1,000    27                                                                    100      19                                                                    10        8                                                                    1         --*                                                    Thiarubrine D 10,000   32                                                                    1,000    26                                                                    100      23                                                                    10       14                                                                    1        --                                                      Thiarubrine E 10,000   50                                                                    1,000    50                                                                    100      36                                                                    10       17                                                                    1        --                                                      ______________________________________                                    

Results were obtained by agar diffusion method. Disc diameter - 6 mm. -* = no zone over disc diameter.

                  TABLE 7                                                          ______________________________________                                         LC.sub.50 (24 hrs.) Data Obtained by Different Method                          Doses Required to Inhibit Growth of C. albicans                                in 50% During 24 hrs. (ng/ml)                                                            Thiarubrines                                                         Organism    A      D      E    Fungizone                                                                              Econazole                               ______________________________________                                         C. albicans 40      7      6      64   56                                      P815 mastocytoma                                                                           81     85     28   50,000                                          ______________________________________                                    

Data on degradation of Thiarubrine D (ThD) expressed as changes of concentration required to inhibit growth of C. albicans in 50% during 24 hrs. exposure (Lc₅₀ 24 hrs.).

                  TABLE 8A                                                         ______________________________________                                         Degradation of ThD under Exposure                                              to "Bench" Cool White Light                                                    Time of Exposure                                                                              LC.sub.50 (24 hrs.) ng/ml                                       ______________________________________                                         24        hrs.            " 5,000                                              6         hrs.            " 5,000                                              0.5       hrs.            140                                                  15        mins.           26                                                   5         mins.           12                                                   0         mins.           7                                                    ______________________________________                                    

                  TABLE 8B                                                         ______________________________________                                         Temperature and Time Dependent Degradation                                     of ThD in Water Solution (Saboraud Dextrose Medium)                                            LC.sub.50 24 Hrs. ng/ml                                                        Temperature                                                    Time of Exposure  37° C.                                                                          Room                                                 ______________________________________                                         24 hrs.           210     110                                                  6 hrs.            85      42                                                   2 hrs.            33      22                                                   0.5 hrs.          20      16                                                   0 hrs.             7       7                                                   ______________________________________                                    

                  TABLE 9                                                          ______________________________________                                         Influence of Exposure of ThD for Two Hrs. to Different                         pH (5, 6, 7, 8) of Sodium Phosphate Buffer                                            pH  LC.sub.50 (ng/ml)                                                   ______________________________________                                                5   40                                                                         6   34                                                                         7   20                                                                         8   52                                                                  ______________________________________                                    

                  TABLE 10                                                         ______________________________________                                         Changes in LC.sub.50 Addition of Fetal Bovine Serum (FBS)                      to Saboraud Dextrose Medium (SAB)                                              Medium          LC.sub.50 (ng/ml)                                              ______________________________________                                         SAB              7                                                             SAB + 5% FBS    14                                                             SAB + 10% FBS   16                                                             ______________________________________                                    

                  TABLE 11                                                         ______________________________________                                         Decomposition of ThD in 95% Ethanol in -20° C.                                 Days LC.sub.50 (ng/ml)                                                  ______________________________________                                                 0    7                                                                        21   17                                                                 ______________________________________                                    

                  TABLE 12                                                         ______________________________________                                         Comparison of Doses of ThD Required to                                         Inhibit Different Numbers of C. albicans Cells                                 Cell Density   LC.sub.50 (ng/ml)                                               ______________________________________                                         10.sup.4 /ml    7                                                              10.sup.5 /ml   16                                                              10.sup.6 /ml   42                                                              ______________________________________                                    

                  TABLE 13                                                         ______________________________________                                         Three Thiarubrines F,G and H Were Isolated                                     and their Effectiveness was Found                                                          Lc.sub.50 24 hrs. ng/ml                                                        Thiarubrine                                                        Organism      F            G     H                                             ______________________________________                                         C. albicans    6            8     4                                            P815 mastocytoma                                                                             140          90    59                                            ______________________________________                                    

                  TABLE 14                                                         ______________________________________                                         Biological Activities of Thiarubrines                                                   A    B      C     D    E   F    G    H                                ______________________________________                                         Conc. in plant                                                                            H      L      --  H    H   L    L    L                              Decomp. product                                                                           T      T      T   T    T   T    T    T                              Stability  L      L      L   H    H   H    H    H                              Anti-Candida                                                                              Y      Y      Y   Y    Y   Y    Y    Y                              Anti-Aspergillus                                                                          Y      Y      Y   Y    Y   Y    Y    ?                              Anti-bacterial                                                                            Y      Y      Y   Y    Y   Y    Y    ?                              ______________________________________                                          Legend                                                                         H = high; L = low; T = thiophene; Y = yes; y = less active; ? = not teste                                                                               

As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims. 

We claim:
 1. An isolated substance having antifungal activity of the formula ##STR14## where R₁ is CH₃ or HOCH₂ ;R₂ is ##STR15## --CHOHCH₂ OH; --CHClCH₂ OH; --CHOHCH₂ Cl; or --CH═CH₂ n is 1; m is 2;, provided that when R₁ is CH₃, then R₂ is not CH═CH₂, and pharmaceutically acceptable salts thereof.
 2. An isolated Thiarubrine D substance of the formula: ##STR16##
 3. An isolated Thiarubrine E substance of the formula: ##STR17##
 4. An isolated Thiarubrine F substance of the formula: ##STR18##
 5. An isolated Thiarubrine G substance of the formula: ##STR19##
 6. An isolated Thiarubrine H substance of the formula: ##STR20##
 7. An antifungal composition, comprising a Thiarubrine substance of the formula: ##STR21## where R₁ is CH₃ or HOCH₂ ;R₂ is ##STR22## --CHOHCH₂ OH; --CHClCH₂ OH; --CHOHCH₂ Cl; or --CH═CH₂ n is 1; m is 2;, provided that when R₁ is CH₃, then R₂ is not CH═CH₂, and pharmaceutically acceptable carrier.
 8. The composition of claim 7, in which the Thiarubrine substance is Thiarubrine D of the formula: ##STR23##
 9. The composition of claim 7, in which the Thiarubrine substance is Thiarubrine E of the formula: ##STR24##
 10. The composition of claim 7, in which the Thiarubrine substance is Thiarubrine F of the formula: ##STR25##
 11. The composition of claim 7, in which the Thiarubrine substance is Thiarubrine G of the formula: ##STR26##
 12. The composition of claim 7, in which the Thiarubrine substance is Thiarubrine H of the formula: ##STR27##
 13. The composition of claim 7, wherein the Thiarubrine substance is present at a concentration of about 1 to 100 nanograms/ml of carrier.
 14. The composition of claim 7, wherein the Thiarubrine substance is present at a concentration of about 10 nanograms/ml of carrier. 